The TWiV team takes on an experimental plant-based poliovirus vaccine, contradictory findings on the efficacy of Flumist, waning protection conferred by Zostavax, and a new adjuvanted subunit zoster vaccine.

On episode #292 of the science show This Week in Virology, Vincent visits Medimmune and speaks with Wade, Matt, Nicole, and Ken about why they work in industry and their daily roles in a biotechnology company.

Infection with influenza virus is known to increase susceptibility to bacterial infections of the respiratory tract. In a mouse model of influenza, increased bacterial colonization was also observed after administration of an infectious, attenuated influenza virus vaccine.

Primary influenza virus infection increases colonization of the human upper and lower respiratory tract with bacteria, including Streptococcus pneumoniae and Staphylococcus aureus. Such infections may lead to complications of influenza, including pneumonia, bacteria in the blood, sinusitis, and ear infections.

One of the vaccines available to prevent influenza is an infectious, attenuated preparation called Flumist. To determine if a vaccine such as Flumist increases susceptibility to bacterial infection, the authors created their own version of the vaccine (illustrated) in which the six RNA segments encoding internal proteins were derived from the A/Puerto Rico/8/34 (H1N1) strain (allowing replication in mice), and the HA and NA proteins were derived from A/Hong Kong/1/68 (H3N2). In addition, mutations were introduced into the viral genome that are important for the safe and protective properties of Flumist. For simplicity we’ll call this virus ‘live attenuated influenza virus’, or LAIV.

Mice were inoculated intranasally with a strain of S. pneumoniae known to colonize the nasopharynx, followed 7 days later by LAIV or wild type influenza virus. Inoculation with either virus similarly increased the bacterial levels in the nasopharynx, and extended the time of colonization from 35 to 57 days. In mice that were given only bacteria and no influenza virus, the inoculated bacteria were cleared beginning 4 days after administration. The more extensive and extended colonization of virus-infected mice was not associated with overt disease.

Administration of LAIV or wild type virus 7 days before bacteria also resulted in excess bacterial growth in mice. Similar results were obtained using S. aureus. Administration of S. pneumoniae up to 28 days after virus also lead to excess bacterial growth, despite clearance of the viruses around 7 days after vaccination.

All mice died when they were vaccinated with wild type influenza virus followed 7 days later by a sublethal dose of a highly invasive strain of S. pneumoniae. In contrast, pretreatment with LAIV lead to no disease or death of any mice.

It is not known if these findings in a mouse model directly apply to humans. However, because Flumist reduces influenza virus replication, it is associated with a decrease in secondary bacterial infections. It is possible that, after administration of LAIV to humans, there is an increase in bacterial colonization of the respiratory tract. Upper respiratory tract symptoms are a known adverse effect of LAIV, and it is possible that these might be related to increased bacterial loads. It is important to emphasize that use of LAIV is not associated with severe upper or lower tract disease.

These findings are important because they show that a mouse model could be used to understand why influenza virus infection leads to increased bacterial colonization of the respiratory tract. It will be important to determine the precise mechanisms by which influenza virus infection, and the associated virus and immune-mediated alteration to the respiratory tract, allows enhanced bacterial colonization. At least one mechanism, which we discussed on episode #62 of This Week in Microbiology, involves the disruption of biofilms, allowing bacteria to enter the bloodstream.

Influenza vaccine effectiveness is assessed each year by the U.S. Influenza Vaccine Effectiveness Network. Patients with acute respiratory infection (ARI) are enrolled in the study; respiratory samples are taken and the presence of influenza virus is determined by polymerase chain reaction.

Data from 1,155 children and adults with ARI during December 3, 2012–January 2, 2013 were collected at five study sites and used to determine that the estimated vaccine effectiveness is 62% (95% confidence intervals = 51%–71%). This number represents the overall effectiveness of seasonal influenza vaccine for preventing laboratory-confirmed influenza virus infection associated with acute respiratory infection.

Of 1,155 children and adults with ARI, 416 (36%) were positive for influenza A or B virus. Of these, 236 (57%) were influenza A virus (all H3N2) and 180 (43%) were influenza B viruses. The immunization rate for influenza cases was 32% and 56% for individuals who did not have influenza virus. The ARI of these patients was likely caused by another agent.

It is known that this season’s influenza vaccine is a good match for the circulating viruses. Why then is vaccine effectiveness only 62%? Although the study does not distinguish between patients who received the inactivated (injected) or the infectious (intranasal) vaccine, most vaccine distributed in the US is the former. Because the inactivated vaccine is treated with formaldehyde and detergents, the viral proteins are not in their native state and do not produce the correct antibodies for optimal protection. Because the intranasal vaccine contains infectious virus, the viral proteins are unaltered and induce antibodies that are more protective. In children less than 7 years old, the intranasal vaccine provides a higher level of protection than the inactivated vaccine. It’s unfortunate that the influenza vaccine needs of the US cannot be met by current supplies of FluMist.

Based on the results of this and similar studies, some argue that it is not worth being immunized against influenza. This reasoning is faulty. Even if you are immunized and subsequently contract influenza, you are likely to be less sick than if you had not received the vaccine. According to CDC:

Influenza vaccination, even with moderate effectiveness, has been shown to reduce illness, antibiotic use, doctor visits, time lost from work, hospitalizations, and deaths.

December 2-8 is National Influenza Vaccination Week. It was established in 2005 by the Centers for Disease Control and Prevention to highlight the importance of continuing immunization throughout the holiday season. This year the push to immunize against flu comes as the disease has begun to increase substantially throughout the United States, as shown in the figure (click the figure for a larger version).

During week 47, 812 of 5,342 (15.2%) of respiratory samples tested positive for influenza virus. Of those isolates that were subtyped, most were either H3N2 or an influenza B virus strain. The 2009 swine-origin H1N1 strain has only been found in one sample so far. Fortunately the H3N2 component of the influenza vaccine for 2012-13 is a good match for the circulating H3N2 strain.

A substantial rise in the number of influenza cases typically does not occur until the end of December in the US. The last time that the disease incidence rose so early was in 2003-04. That was one of the most lethal seasons in 35 years, with 48,000 deaths. This year two children have already died of influenza in the US.

The good news is that 112 million doses of influenza vaccine have already been administered this year, and there is still time to be immunized. The CDC recommends that everyone over 6 months of age be immunized against influenza. Vaccination is especially important for individuals who are at risk for developing serious influenza-related complications: pregnant women, children younger than 5 years old (but those less than 6 months of age should not be immunized), people with with chronic medical conditions such as asthma, diabetes, and heart disease, and those over 65 years old.

The influenza vaccine is available in two types: an injected, inactivated preparation, and an infectious version that is sprayed in the nose. After immunization, approximately two weeks are required to be fully protected against infection.

On episode #209 of the science show This Week in Virology, Vincent, Dickson, Alan, and Kathy answer listener email about deformed wing virus, West Nile virus, FluMist, influenza in Canada, viruses and the tree of life, and more.

The suggestion that yearly immunization against influenza might make children more susceptible to serious disease during a pandemic has generated some controversy. Does this idea have merit?

If you have read “Being older is a good defense against 2009 H1N1 influenza”, you are familiar with the concept of ‘heterosubtypic immunity’. After natural infection with influenza virus, the host produces T and B cells directed against internal proteins of the virions. These viral proteins are more conserved among different strains than the surface glycoproteins HA and NA. Upon infection with a different subtype – which occurs during a pandemic – heterosubtypic immunity could limit virus replication and reduce disease and death.

Evidence for heterosubtypic immunity to influenza virus comes mainly from studies in guinea pigs and mice. It has been suggested that this type of immunity could explain why older people appear to be less susceptible to infection with the 2009 H1N1 influenza virus.

What does heterosubtypic immunity have to do with vaccinating children against influenza? Immunization of children against influenza is a good idea, because it reduces the amount of disease. But immunized children don’t develop heterosubtypic immunity – it’s not induced by the vaccine, and natural infection is prevented. This could put children at greater risk for more serious disease when pandemic strains emerge. Absence of heterosubtypic immunity might explain why young children are more likely to develop severe disease after infection with the 2009 H1N1 influenza virus.

Is there a solution to this potential problem? One possibility is more widespread use of infectious, attenuated influenza vaccines such as Flumist, which are known to induce heterosubtypic immunity. New vaccines that induce heterosubtypic immunity are under development. These consist of purified internal virion proteins, such as M2 protein, that undergo less antigenic variation than HA or NA.

Should we stop immunizing young children against influenza, and wait for the development of better vaccines? Of course not! Influenza in children can be serious. We should not allow this preventable disease to occur based on an unproven theory that children might be better off in a future pandemic. Neither do the authors of the hypothesis advocate cessation of immunization:

More research is needed to find out if heterosubtypic immunity contributes to protection against infection with pandemic strains in people and if yearly vaccination against seasonal influenza prevents the induction of heterosubtypic immunity. The development and use of vaccines that can induce broad protective immunity might be a solution for these potential problems and we think this is a priority.

The results of animal experiments do not dictate immunization policy. But what we learn from animal experiments often lead to hypothesis which can be tested by studies in humans.

There have been many interesting responses to my recent post, “Are you receiving the influenza 2009 H1N1 vaccine?” Some individuals have already been immunized or plan to do so shortly. Others are concerned about the safety and efficacy of the monovalent preparations. As pointed out recently in a Nature editorial, “Mass-vaccination campaigns…must take public concerns into account”, and “officials should focus on providing people with the information they need to make good choices for themselves.” Here are some facts about the influenza H1N1 vaccine for those who haven’t yet made up their minds whether or not to be immunized.

Four companies are licensed to produce the 2009 H1N1 influenza vaccine for the US – CSL Limited, Novartis Vaccines, Sanofi-Pasteur Inc., and MedImmune. The US Food & Drug Administration has published on their website the package insert for each product. These are multipage documents with a good deal of information, including indications, dosage, contraindications, adverse reactions, and the results of clinical studies. Links to the package insert for each vaccine are listed below.

For example, the package insert for Afluria indicates that the vaccine is supplied as a sterile suspension for intramuscular injection in two forms: a 0.5 mL preservative-free, single-dose, pre-filled syringe; and a 5 mL multi-dose vial containing ten doses. Thimerosal, a mercury derivative, is added as a preservative in the multi-dose vial; each 0.5 mL dose contains 24.5 micrograms of mercury.

The results of two clinical studies on the safety and efficacy of Afluria are also reported: a US study of 1,357 individuals, and a UK study of 275 subjects. No serious adverse effects were reported in either study. The seroconversion rates are included in the form of hemagglutination-inhibition titers.

I encourage everyone to read at least one of these package inserts to become familiar with the extent to which these vaccines have been tested in people. If any aspects of these documents are not clear, don’t hesitate to contact me.

Note added after publication: It was pointed out in the comments that the safety and efficacy data reported in the package inserts were obtained with the seasonal influenza vaccines, Afluria, Fluvirin, Fluzone, and Flumist, not the 2009 monovalent H1N1 vaccine. My apologies for implying otherwise. I will locate the safety and efficacy data for the monovalent H1N1 vaccines and update the post with that information.

Second addendum: According to the FDA, the influenza A (H1N1) 2009 monovalent vaccine has been tested in the same way as new seasonal vaccines that are produced each year. The inactivated influenza A (H1N1) monovalent vaccine manufactured by CSL Limited was tested in 120 adults. By day 21 after vaccination, HI antibody titers of 1:40 or more were observed in 116 (97%) of 120 adults who received a 15 μg dose. Injection site tenderness or pain was reported by 46% of subjects, while 45% of the subjects reported one or more systemic symptoms (headache, malaise, or myalgia). These observations are similar to those observed in testing of seasonal influenza vaccines. The preliminary results have been published.

The Sanofi Pasteur 2009 H1N1 vaccine has been tested in children aged 6-35 months, 3-9 years, and 10-17 years, and the results have been reported by NIAID. After immunization with a single 15 μg dose of vaccine, 25%, 36% and 76% of individuals in each age group developed HI antibody titers of 1:40 or more. No information on adverse effects have been provided.

Medimmune and Novartis have reported immunogenicity and safety study results similar to those observed for seasonal vaccines, but the data have not yet been released.

Although the 2009 H1N1 vaccines produced by all four manufacturers have been approved by the FDA, all the data used for approval have not yet been published or released. As soon as these data are available I will pass them on.

A new type of vaccine against influenza, made with virus-like particles, has been shown to protect ferrets from infection with the 2009 H1N1 swine-origin strain. What is a virus-like particle, and how is it produced?

If you have been taking influenza 101, you know that new virus particles are produced in infected cells by budding. During this process, the membrane bulges from the cell and is eventually pinched off to form a free particle. These virus particles contain the viral RNA segments, and an assortment of viral proteins including PA, PB1, PB2, NP, M1, M2, HA, and NA. But not all of those viral proteins are needed to produce an influenza virus particle. When only the viral HA, NA, and M1 proteins are synthesized in cells, particles are released from cells that look very much like influenza virions (illustrated). These are called ‘virus-like particles’ because they resemble influenza viruses, but lack the viral genome and many viral proteins.

Influenza virus-like particles are not infectious, but they are immunogenic: when injected into animals, they induce the production of anti-viral antibodies that can block infection. In one study, virus-like particles were produced in cultured insect cells by using an insect virus vector – a baculovirus – to deliver genes encoding the influenza HA, NA, and M1 proteins. Mice inoculated with these virus-like particles were protected after challenge with infectious virus. More recently, mice vaccinated with virus-like particles produced with proteins from an H5N1 avian strain were protected against challenge with lethal H5N1 viruses.

These findings suggest that virus-like particles could be used in humans to protect against influenza infection. They offer a number of advantages over the current influenza virus vaccines, most of which are prepared by growing virus in embryonated chicken eggs. Viral infectivity is destroyed with formalin, and the virions are then disrupted with detergents. Virus-like particle vaccines would not require these treatments, and would be available to individuals with egg allergies. Some influenza virus vaccines are produced in cell culture, but these are also treated to eliminate infectivity.

Another important advantage of the virus-like particle vaccine is that it can be produced relatively rapidly: within weeks, compared to months for egg-produced vaccines. This property would be especially useful when new pandemic strains emerge. For example, the swine-origin H1N1 influenza virus emerged in the spring of 2009, and vaccine manufacturers are scrambling to have a product ready for the fall.

Because an influenza virus-like particle vaccine is a new type of vaccine, many years of testing in animals and in humans will be required before it can be used. Some of the questions that must be addressed include the safety of the vaccine in humans, whether the anti-viral antibody repertoire induced by the vaccine is sufficiently broad, and of course whether immunization confers efficient protection against challenge in the majority of recipients.

I am particularly curious about how an influenza virus-like particle vaccine would compare with the infectious, attenuated influenza vaccine, Flumist. This intranasally-administered vaccine mimics a natural infection and has been shown to be more effective in preventing influenza than inactivated vaccine. While virus-like particle vaccines are an attractive option, they are not infectious and therefore might not induce the same antibody repertoire as would an infectious virus. A disadvantage of Flumist is that it is produced in eggs. If influenza virus-like particles prove safe and efficacious in humans, they could replace the egg-grown, inactivated vaccines.